• Overall, evidence from January 2000 to March 2024 was inconclusive across populations to determine the average daily dietary protein and indispensable amino acid requirements across populations.
• For most populations, one or two studies of higher methodological rigor examined protein or indispensable amino acid requirement estimates. When more than two studies were available, different designs and/or methods to calculate the estimate requirements were used, making comparisons between studies difficult.
• We identified a critical need for the development of a single standardized and validated method to assess protein and amino acid requirement estimates. Future studies, using this new method, should focus on population groups including infants, children, adolescents, pregnant people, and lactating people where protein intake is essential for appropriate growth and development.

Objective: This review assesses the evidence on requirements for average daily dietary protein and individual indispensable amino acid intake for healthy individuals by life stage and sex. The results will inform future updates to the Dietary Reference Intakes (DRI) for protein.

Data sources: We searched Medline, EMBASE, AGRICOLA, and Scopus from January 2000 through March 2024 for studies from peer-reviewed published literature and supplemented with citation searching of relevant systematic reviews and original research.

Review methods: Two reviewers independently screened titles, abstracts, and full-text publications. We included randomized and nonrandomized controlled trials, prospective cohort, and nested case-control studies that enrolled infants through older adults and investigated total protein and amino acid requirements using a variety of methods (i.e., nitrogen balance, indicator amino acid oxidation, etc.) We extracted data, assessed risk of bias, synthesized results from low to moderate risk of bias studies in a narrative manner, and evaluated the strength of the evidence supporting the conclusions. The protocol was registered on PROSPERO (CRD42023446618).

Results: We identified 11,344 studies of which 68 articles reporting on 66 unique studies were eligible for the review and 45 studies were assessed as low or moderate risk of bias. For most populations, one or two studies of higher methodological rigor were available for both protein and amino acid requirements. For infants, six studies examined requirements for isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. For children and adolescents, seven studies examined requirements for protein, lysine, methionine, phenylalanine, and total branched chain amino acids. For pregnant people, four studies examined requirements for protein, lysine, and phenylalanine. For adults aged 19-50 years, 16 studies examined requirements for protein, leucine, lysine, methionine, phenylalanine, threonine, and valine and for adults aged 51->70 years, six studies examined requirements for protein, leucine, and phenylalanine. Both males and females were studied for all requirements except pregnant people (females only), valine requirement estimates for infants (males only), phenylalanine requirements for children and adolescents (not reported), and protein, leucine, methionine, phenylalanine, threonine, and valine requirements for adults aged 19-50 years (males only). Commonly used methods included indicator amino acid oxidation, 24-hour indicator amino acid oxidation, 24-hour indicator amino acid balance, and nitrogen balance.

Conclusions: Overall, evidence from January 2000 to March 2024 is inconclusive across populations to determine the average daily dietary protein and indispensable amino acid requirements.